Rapid small volume detection of blood ammonia

ABSTRACT

A method for measuring ammonia in a blood sample may involve positioning the blood sample in proximity with an ammonia gas sensor, generating a current with the ammonia gas sensor in response to ammonia gas released from the blood sample, and measuring the current generated by the ammonia gas sensor, using a current measurement member coupled with the ammonia gas sensor. A device for measuring an ammonia level in a blood sample may include a blood sample containment member, an ammonia gas sensor coupled with the blood sample containment member, and a current measurement member coupled with the ammonia gas sensor. The method and device may be used to measure an ammonia level in a blood sample as small as one drop of blood, or approximately 0.05 mL of blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/938,467, entitled “Rapid Small Volume Detection of BloodAmmonia,” filed on Feb. 11, 2014. The full disclosure of theabove-listed patent application is hereby incorporated by referenceherein.

BACKGROUND

Hyperammonemia is a metabolic disturbance characterized by excessammonia in the blood. It is a dangerous condition that may lead to braindysfunction (encephalopathy) and even death. Some cases of sudden infantdeath syndrome (SIDS) have been linked to undiagnosed metabolicdisorders that caused hyperammonemia. A number of different conditionsmay cause hyperammonemia, such as inherited genetic mutations, liverdamage, blind loop syndrome associated with gastrointestinal surgery,inflammatory bowel disease and some types of chemotherapy medicationsused for cancer treatment. Hyperammonemia is typically simple to treatif detected early enough. Unfortunately, there are no currentlyavailable, simple, quick tests for detecting hyperammonemia. This meansthat many cases of hyperammonemia go undetected until it is too late,often resulting in patient death.

Conditions that cause hyperammonemia may be inherited or acquired. Manydifferent inherited genetic mutations may cause hyperammonemia. Ammoniais converted to the less toxic substance urea prior to excretion inurine by the kidneys. Some children have genetic mutations that disruptthe urea cycle in the body. These children require lifetime monitoringof blood ammonia levels. Acquired causes of hyperammonemia, includingthose mentioned above, also require long term monitoring of bloodammonia levels. Unfortunately, there is no convenient way for patientsto monitor their blood ammonia levels on an ongoing basis.

Sudden infant death syndrome is the largest single cause of death inchildren in the industrialized world. A small but significant fractionof SIDS deaths are due to an unrecognized metabolic disorder associatedwith hyperammonemia. Many of these disorders become symptomatic eitherin the newborn or in the first few months of life. Indeed, some statesperform neonatal tests for genetic diseases, such as fatty acidoxidation disorders or organic acidemias, which are associated withhyperammonemia. Rather than measuring blood ammonia levels directly, thetests employ tandem mass spectrometry to detect specific metabolitesthat accumulate in these conditions. The tests must be performed from adrop of blood obtained by a heel stick of the newborn infant, becausevenipuncture is technically difficult, morbid, and invasive. Currently,the United States screens for only a small number ofhyperammonemia-associated defects. Other economically advanced countriesoffer few screening tests of any kind.

Although hyperammonemia damages the brain, it can be effectively andsimply treated if detected early enough. One simple treatment involvesadministering lactulose to the patient. Lactulose is a non-absorbedsugar, which acidifies the stool and thus prevents absorption of ammoniainto the blood from the intestine. Another treatment involvesadministering the antibiotic Rifaxamin, which kills urea-producingbacteria in the intestine. These treatments are simple, inexpensive andeffective, if used early enough in the course of hyperammonemia.

The most pressing issue in detecting and treating hyperammonemia lies indetection. All of the currently available detection methods requireprocessing blood to acquire plasma, and then testing the plasma forammonia. Processing involves spinning the blood in a centrifuge toseparate the plasma from the red blood cells. This must be done in a labthat has centrifuge equipment. The plasma must then be quickly tested,as soon as possible after blood processing. In most cases, the bloodprocessing and ammonia level measurement must be carried out in twodifferent areas of the hospital. Thus, a patient must go to ablood-processing lab attached to a hospital to have blood taken for thetest. Additionally, the current tests for blood ammonia require anamount of blood that can only be acquired by venipuncture. This makestesting infants and small children additionally challenging.

The most commonly used clinical laboratory test for measuring plasmaammonia utilizes the glutamate dehydrogenase reaction, in which ammoniumion reacts with 2-oxoglutarate and NADPH to form glutamate, NADP andwater. Absorbance spectroscopy at 340 nm measures the decrease in NADPH.The assay is highly specific and effective over a broad range of ammoniaconcentrations. However, the procedure is lengthy and complex, requiringvenipuncture for several milliliters of blood, followed bycentrifugation for plasma. Samples must be transported on ice to acentral laboratory to minimize false elevation of ammonia levels fromglutamine deamination in the blood cells. Thus, the standard clinicaltest is incompatible with close monitoring of patients at risk forhyperammonemia.

One alternative to the standard plasma ammonia analysis is to measurethe ammonia concentration in whole blood. To avoid interference fromother blood components, this strategy liberates ammonia in gaseous formfor separate quantification. Gas-sensing electrodes detect ammonia byrelease of ammonia by alkalization of the whole blood sample. Theelectrode is placed above the surface of the sample and protected by agas permeable membrane. This method has two major disadvantages,however—it requires a large amount of blood to work, and it takes a longtime to analyze one sample—10 to 15 minutes at low ammonia levels.

Colorimetric reactions may also be used measure ammonia by spectrometry.Such reactions include the indophenol reaction, which generates a bluecolor, and the Nessler reaction, which generates a brown-orange color.The disadvantage of such methods is that other substances in the blood,such as amino acids and glutamine, can affect the reactions, leading toinaccuracy.

A more recent colorimetric method for measuring ammonia in whole bloodhas been developed and incorporated into a device known as the BloodAmmonia Checker or, in a more recent version, the PocketChem BloodAmmonia Analyzer (available from Woodley Equipment Company Ltd.). Tomeasure ammonia, a small drop of blood is placed on a test strip thatcontains alkaline salts that liberate ammonia from the blood. Theammonia diffuses through a porous separator to a color-developing layercontaining bromocresol green. The ammonia is quantified by colorimetryafter 3-4 minutes. The problem with this device is that it has beenshown to have problems with accuracy. Additionally, sensitivity fordetecting elevated ammonia levels is questionable. The dynamic range ofa dye-based assay is necessarily limited by the pKa of the dye and thesensitivity of the color detection system. Finally, the price of theBlood Ammonia Analyzer is $3,000.

Therefore, it would be very desirable to have an improved method anddevice for testing blood ammonia levels. Ideally, such a method anddevice would be simple to use, relatively inexpensive, and portable.Also ideally, the method and device would be convenient for use in ahospital, physician's office or patient's home. Finally, it would beideal if such a method and device could be used for one-time bloodammonia level testing, ongoing monitoring of blood ammonia levels, orboth. At least some of these objectives will be met by the embodimentsdescribed below.

BRIEF SUMMARY

The present disclosure describes a device that uses an electrochemicalreaction to detect gaseous ammonia liberated from blood. Variousembodiments of the device include a blood sample containment member, anammonia gas sensor, and a current measurement member coupled with thegas sensor. A small volume blood sample, which in some cases may be assmall as a drop of blood (approximately 0.05 mL), is placed in or on theblood sample containment member, which may be a container or substrate.Ammonia liberated out of the blood sample then generates a current inthe ammonia gas sensor, which in some embodiments may be an ammonia fuelcell, and this current enters the current measurement member (anelectric circuit, for example) Measured current may then be displayed onan ammonia level display. Electrochemical detection should provide moreaccurate results than the dye-based tests in current use, because itdoes not share the limited dynamic range of a dye-based assay.Furthermore, miniature ammonia fuel cells, which may be used as theammonia gas sensor in one embodiment, are inexpensive and readilyavailable. For example, ammonia fuel cells used for industrial safetyapplications cost approximately $200 each. (One example of a currentlyavailable ammonia fuel cell is pictured in FIG. 1.)

Methods described herein typically involve placing a blood sample in oron a sample container or substrate in proximity to an ammonia gassensor, allowing ammonia liberated from the blood sample to generate acurrent in the ammonia gas sensor, and measuring the current with acurrent measurement member coupled with the ammonia gas sensor. Themethod also typically involves providing a blood ammonia level to a uservia a display member coupled with the ammonia gas sensor and/or thecurrent measurement member. In some embodiments, the method may alsoinvolve mixing an alkaline substance with the blood sample to facilitateand/or hasten the ammonia leaving the blood. For example, one suchsubstance may be K2CO3 (potassium carbonate), while another example maybe a solution of LiCl/LiOH (Lithium Chloride/Lithium Hydroxide). In somecases, such a solution may be mixed with the blood sample, and themixture may be stirred or agitated. In other embodiments, the bloodsample may simply be placed on a solid support in proximity with thefuel cell, without any further treatment of the sample.

The device embodiments described herein may be very small, portable,inexpensive, and quick to use, thus allowing for blood ammonia testingat a patient's bedside (hospital, nursing home or the like), in aphysician's office, at home, or wherever is most convenient to apatient. In some embodiments, for example, the ammonia testing devicemay be as small and convenient to use as a blood glucose monitoringdevice commonly used by diabetics. In other embodiments, the device mayinclude additional features, such as capability for transmitting ammonialevels directly to the patient's electronic medical record when testsare performed in the hospital setting. The devices and methods describedherein for rapid small volume detection of blood ammonia may provide asignificant improvement in detecting and monitoring hyperammonemia.Thus, they may be used to screen millions of patients worldwide to helpprevent brain damage and death.

In one aspect, a method for measuring ammonia in a blood sample mayinvolve positioning the blood sample in proximity with an ammonia gassensor, such as an ammonia fuel cell, and measuring current generated bythe fuel cell in response to ammonia released from the blood sample. Themethod may also involve displaying the measured current on a displaycoupled with the current measurement member. In some embodiments,positioning the blood sample may involve forming a sealed chamber (or“compartment”), in which the ammonia released from the blood has accessto an anode end of the fuel cell. Some embodiments may further includemixing the blood sample with an alkaline substance. For example, in twoalternative embodiments, the alkaline substance may be potassiumcarbonate or an aqueous solution of lithium chloride and lithiumhydroxide. Optionally, the method may further include stirring oragitating the blood/alkaline substance mixture.

As mentioned above, in some embodiments, the blood sample may be assmall as a drop of blood, or no more than approximately 0.05 mL ofblood. In some embodiments, positioning the blood sample may involvecontacting a sealed container with the ammonia gas sensor, such that ananode end of the gas sensor is exposed to an open space in the containerthat contains the blood sample. In other embodiments, positioning theblood sample may involve positioning a substrate holding the bloodsample in proximity with an anode end of the ammonia gas sensor, wherethe anode end and the blood sample are located within a sealedcompartment after the positioning step. In some embodiments, an ammeteris used to measure the current generated by the fuel cell.

In another aspect, a method for measuring ammonia in a blood sample mayinvolve positioning the blood sample in proximity with an ammonia fuelcell or other ammonia gas sensor, generating a current with the ammoniafuel cell or other sensor in response to ammonia released from the bloodsample, and measuring the current generated by the fuel cell or othersensor.

In another aspect, a device for measuring an ammonia level in a bloodsample may include a blood sample containment member, an ammonia gassensors, such as an ammonia fuel cell, coupled with the blood samplecontainment member, and a current measurement member coupled with thegas sensor. In some embodiments, the blood sample containment member maybe removably attachable to the gas sensor to form a sealed compartmenthousing an anode end of the fuel cell and the blood sample. In someembodiments, the blood sample containment member comprises a substratefor holding the blood sample. Optionally, such an embodiment may includea substrate that acts as a base to release ammonia, or a substratetreated with substance (for example, a base and/or a salt). The bloodsample would then be applied to the substrate.

Typically, the device will be small enough to be easily portable. Thedevice will also be configured to measure blood ammonia levels usingonly a very small sample of blood, such as no more than a drop of blood,or no more than approximately 0.05 mL of blood. In some embodiments, theammonia fuel cell may be a miniature ammonia fuel cell, similar to thoseused in various industrial applications. In some embodiments, thecurrent measurement member may be an ammeter or a potentiostat. In someembodiments, the blood sample containment member may form a chamber influid communication with an anode end of the ammonia gas sensor, and thecurrent measurement member may be an electric circuit coupled with theanode end and a cathode end of the ammonia gas sensor.

In some embodiments, the device may further include a housing forcontaining the blood sample containment member, the ammonia gas sensorand/or the current measurement member. In some embodiments, the devicemay further include a display on the housing for displaying the measuredblood ammonia level. The display may be configured to display themeasured blood ammonia level numerically, as a linear display (such as agraph), and/or via any other suitable representation, according tovarious embodiments.

These and other aspects and embodiments will be described in furtherdetail below, in reference to the attached drawing figures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of an industrial ammonia fuel cell;

FIG. 2 is a diagrammatic representation of a blood ammonia levelmeasurement device, according to one embodiment;

FIG. 3 is a graph showing time on the horizontal axis and current on thevertical axis, illustrating a current vs. time response to ammoniarelease from blood, according to one embodiment and one sample;

FIG. 4 is a graph illustrating a standard curve measurement of ammoniain whole blood, using devices and methods according to one embodiment;and

FIG. 5 is graph illustrating a conventional plasma assay vs. a wholeblood ammonia level detection technique, according to one embodiment.

DETAILED DESCRIPTION

The following description is directed to embodiments of a device andmethod for measuring ammonia levels in a blood sample. The embodimentsprovided below are meant to be exemplary in nature and should not beinterpreted as limiting the scope of the invention. For example, invarious alternative embodiments, it may be possible to eliminate oralter one or more device features, eliminate or alter one or more methodsteps, change the order of method steps, and/or the like. As onespecific example, although the following description focuses on the useof the device and method embodiments for detecting ammonia levels inblood samples, in alternative embodiments, these same embodiments orvariations thereof may be used to detect ammonia levels in othersubstances.

Referring now to FIG. 2, in one embodiment, a rapid, small volume, bloodammonia detection device 10 includes a blood sample containment member12 coupled with an ammonia (NH3) fuel cell 14, which is coupled with acurrent measurement member 16. In various alternative embodiments, fuelcell 14 may be replaced by any other suitable ammonia gas sensor, suchas but not limited to a redox mediator. Therefore, although theembodiment illustrated in FIG. 2 and much of the following descriptiondiscusses a “fuel cell,” alternative embodiments may include anysuitable substitute ammonia gas sensor. Therefore, this applicationshould not be interpreted as being limited to only embodiments includingfuel cells.

Similarly, in one embodiment, as shown in FIG. 2, blood samplecontainment member 12 may be a container for holding a blood sample. Oneexample of blood containment member 12 may be a vial with a cap and amembrane. In this embodiment, a blood sample may be placed in the vial,and the cap and membrane may seal containment member 12. Whencontainment member 12 is then attached to fuel cell 14 and/or a housingcontaining fuel cell 14, the membrane may be pierced, thus forming achamber that is exposed to fuel cell 14 but is still sealed. Inalternative embodiments, blood sample containment member 12 may be asubstrate, such as a plate, slide, piece of flat material or the like.In substrate embodiments, blood sample containment member 12 may bepositioned in proximity to fuel cell 14 for measurement of the ammoniagas. In the embodiment shown, current measurement member 16 is apotentiostat. In alternative embodiments, current measurement member 16may be an ammeter or other type of circuit or other current measurementdevice.

Containment member 12, fuel cell 14 and current measurement member 16may be entirely or partially contained in a housing 18 a, 18 b. In thediagrammatic representation of FIG. 2, housing 18 a, 18 b is depicted ashaving a blood sample portion 18 a and a fuel cell portion 18 b, whichmay fit together to form a seal, thus sealing in the blood sample withinan airtight chamber. (Alternatively, blood sample portion 18 a maysimply be blood containment member 12—in other words, such an embodimentwould not have a separate blood sample housing portion 18 a.) In thisconfiguration, openings in containment member 12 and blood sampleportion 18 a allow an anode end (or “anode side”) of fuel cell 14 tohave access to what is referred to herein as a headspace 20—the spacedirectly above the blood sample and in air contact with the anode end offuel cell 14. Otherwise, the blood sample and anode end are sealed offfrom outside air. As illustrated in FIG. 2, in some embodiments, currentmeasurement member 16 may extend out of fuel cell portion 18 b of thehousing. In an alternative embodiment, containment member 12, fuel cell14 and current measurement member 16 may all be housed within housing 18a, 18 b. In another alternative embodiment, the housing may be aone-piece structure, and blood sample containment member 12 may beinserted into it once a blood sample is loaded, or a blood sample itselfmay be inserted into device 10 through an opening. Optionally, a displayon an outside of housing 18 a, 18 b (not shown) may display a measuredammonia level. In some embodiments, such as the one depicteddiagrammatically in FIG. 2, containment member 12 may actually be itsown housing 18 a.

In various embodiments, containment member 12 may have any suitablesize, shape and configuration and may be made of any suitable material.For example, containment member 12 may be a cuplike container, with ameans for sealing the open end of the cup to fuel cell 14.Alternatively, containment member 12 may be a vial with a sealed orsealable cap. A blood sample may be inserted into the vial or cup, andin one embodiment device 10 may include a puncturing element thatpunctures one or more holes in a cap of the containment member 12 whenit is attached to device 10, thus allowing liberated blood ammonia tocontact fuel cell 14. In some embodiments, containment member 12 may beextremely small, since it typically only needs to hold one drop or 50microliters of blood. As described further below, for example, in someembodiments containment member 12 may simply be a flat substrate.

Fuel cell 14 may be any currently available or not-yet-invented ammoniafuel cell, such as but not limited to currently available, small,lightweight ammonia fuel cells that are worn as badges by industrialworkers at risk for ammonia exposure. As mentioned above, in otheralternative embodiments, fuel cell 14 may be replaced by an alternativetype of ammonia gas sensor. Fuel cell 14 may include three electrodes,one of which attaches to an anode of current measurement member 16, oneof which attaches to a cathode of current measurement member 16, and oneof which attaches to a reference electrode. In one embodiment, all threeof these attachments are wired attachments. Other electrodeconfigurations may be possible in alternative embodiments. For example,it may be possible to eliminate or substitute some other configurationfor the reference electrode.

Device 10 is configured to detect gaseous NH3 liberated from a bloodsample and exposed to fuel cell 14 in headspace 20, thus generating acurrent response proportional to the NH3 concentration in the sample.During operation, sample containment member 12 is sealed to the anodeside of fuel cell 14. The blood sample is injected into containmentmember 12 and subsequently treated with an alkaline substance, such asbut not limited to potassium carbonate, LiCl/LiOH, other hydroxides, andother salts. The increase in the pH and ionic strength of the blood whenmixed with a substance like potassium carbonate or LiCl/LiOH causes therelease of NH3 into headspace 20. NH3 oxidation at the anode end of fuelcell 14, coupled with oxygen (O2) reduction at the cathode end of fuelcell 14, generates a current that is measured by current measurementmember 16. Since release of NH3 into headspace 20 is faster than itsconsumption at the anode, NH3 effectively equilibrates between headspace20 and the blood sample. The current rises to a plateau value that isproportional to this steady state headspace concentration, and thereforeproportional to the sample concentration. The response is taken as thepeak current, which provides an accurate measurement of blood ammonialevel.

The amount of blood used for the sample may be very small. For example,in some embodiments, one drop of blood may be used—e.g., no more thanapproximately 0.05 mL (50 microliters) of blood. This is in contrast tocurrently available blood ammonia measurement techniques, whichtypically require at least 3000-10,000 microliters of blood. In variousembodiments, any suitable alkaline solution may be mixed with the blood,or in other alternative embodiments, blood may be measured withoutmixing with an alkaline solution. In one embodiment, it was found to beadvantageous to elevate the pH of the blood sample to approximately 11,using LiOH, while at the same time increasing the ionic strength of theblood sample using LiCl. In an alternative embodiment, it was found tobe advantageous to use potassium carbonate (K2CO3). In general,carbonates, such as potassium carbonate, may be preferable to hydroxidesfor safety reasons.

In some embodiments, the blood sample may be injected, or otherwisedelivered into containment member 12. In alternative embodiments, it maybe possible to simply apply a drop of blood to a solid substrate, suchas a flat piece of glass, glass fibers, paper or other material. Theflat substrate may be placed immediately below the anode end of fuelcell 14. In various alternative embodiments, the substrate may be eitheruntreated or pretreated with a substance, such as a salt and/or base. Insome embodiments, device 10 may include a finger stick component, suchthat a patient may prick his or her finger, a drop of blood from thefinger deposits on the sample substrate, and device 10 detects the bloodammonia level. Detection of the ammonia level in the blood sample bydevice 10 may in some embodiments occur very quickly, for example in afew minutes or even just a few seconds.

Device 10 may provide the measurement of the blood ammonia level in anumber of different ways in different embodiments. For example, in someembodiments, device 10 may include a built-in, digital, numericaldisplay, which shows a measured ammonia level. In such an embodiment,device 10 may optionally beep, flash or otherwise signal when theequilibrium ammonia level is reached, analogous to digital thermometersthat signal to a user when the body temperature is measured. In otherembodiments, device 10 may store and/or transmit blood ammoniameasurement for display on another device, such as a laptop or desktopcomputer. For example, device 10 may have Bluetooth capabilities or mayplug into a computer via a USB port.

In some embodiments, device 10 may be entirely disposable, while inalternative embodiments, some or all of device may be reusable. Forexample, in some embodiments, containment member 12 may be disposablewhile the remainder of device 10 is reusable. In other embodiments, allof device 10 may be reusable, and a blood sample carrying substrate maybe placed into containment member 12, such that the substrate isdisposed of after use and the container is reused.

Experimental Measurement of NH3 in Whole Blood

Experiment #1

An initial set of experiments was performed to optimize the release ofNH3 from blood and determine whether device 10 responds linearly to NH3content in blood. (Anonymous whole blood samples were obtained from theStanford Blood Bank according to their human subjects protocol.) Rapidresponses were obtained by injecting 300 μL of whole blood into samplecontainment member 12, followed by 180 μL of an aqueous solutioncontaining 0.2 M LiOH and 12 M LiCl (“LiOH/LiCl”). The blood andreagents were mixed with a magnetic stir bar while the current responsefrom the detector was recorded. FIG. 3 shows one example of a typicalcurrent vs. time trace for this experiment. Device 10 reached aquasi-steady state current within 3 minutes of the addition ofLiOH/LiCl. Unless otherwise noted, this optimized procedure was used forall whole blood analyses discussed below.

Experiment #2

A set of blood samples spanning a range of NH3 concentrations wasprepared by dividing blood from a single donor into 5 aliquots andadding a different amount of NH3 in the form of NH4Cl to each aliquot.The amount of NH3 added ranged from 0 to 800 μM. FIG. 4 shows theresponse of the instrument as a function of the amount of added NH3. Asmooth linear response was obtained across the 5 samples with a slope of1 nA/μM NH3. As expected, an appreciable response was obtained for thesample with no added NH3, which reflected the amount of NH3 naturallypresent in the sample. This experiment demonstrated the ability ofdevice 10 to detect NH3 directly from whole blood and quantify thedifference in NH3 content between samples.

Experiment #3

Ideally, whole blood analysis of ammonia levels, using the devices andmethods described herein, will strongly correlate with NH3 measured forplasma. To address this question, response of device 10 to the NH3released from whole blood samples was compared to the NH3 concentrationin the plasma of the same samples measured by the conventional enzymaticanalysis. Fresh 10 mL whole blood samples were obtained from 5 healthydonors at the Stanford Blood Center and transported on ice to the lab.The samples were analyzed in series to minimize the time between wholeblood and plasma analysis for each individual sample. An aliquot wasremoved from the first sample and kept on ice, while the remainder ofthe sample was centrifuged to separate the plasma. Duringcentrifugation, the whole blood aliquot was analyzed in duplicate withthe instrument. The plasma that had been separated was then assayed intriplicate using the conventional enzymatic assay. This procedure wasrepeated for the 4 remaining whole blood samples. FIG. 5 shows a plot ofthe instrument response to the whole blood aliquots vs. the NH3concentration in the corresponding plasma. A good linear fit (R2=0.90)was obtained for the six samples, which had measured plasma NH3 levelsranging from 52 μM to 77 μM. Furthermore, device 10 response was lessnoisy than the conventional enzyme-based plasma assay. Thus, the datasuggest that device 10 is potentially much more reliable than both theconventional enzyme-based plasma assay and the dye-based Blood AmmoniaChecker.

Measurement of NH3 from a Drop of Blood Spotted onto a Solid Support

Especially for home detection of blood ammonia, it would be ideal torequire only the smallest possible blood sample volume and to make theanalysis procedure as simple as possible. Ideally, the analysis could beperformed with a drop (20-50 μL) of blood on a solid support. In somecases, the support may be preloaded with salt and/or base that wouldfacilitate release of NH3 from the blood sample as it dries. Apreliminary experiment to assess the feasibility of this approach wasperformed.

A fresh whole blood sample was divided into two aliquots, and 167 μM ofNH4Cl was added to one aliquot. Glass microfiber discs were soaked witheither KCl solution or KCl solution+LiOH/LiCl, then rinsed with H2O anddried. The disc was placed into sample-mixing containment member 12 ofdevice 10. A 50 μL drop of blood was added to the disc, and samplecontainment member 12 was immediately sealed to fuel cell 14. Table 1shows the current response for blood drops with and without added NH4Cl,using the two methods for pre-treating the glass fiber filter, eitherwith KCl alone or KCl plus LiOH.

TABLE 1 Whole blood spotted onto a solid support Response Blood sampleMicrofiber filter treatment (nA) None Soaked with KCl solution, dried 11Native sample Soaked with KCl solution, dried 28 +167 μM NH4Cl Soakedwith KCl solution, dried 44 Native sample Soaked with KCl + LiOH/LiClsolution, 22 dried +167 μM NH4Cl Soaked with KCl + LiOH/LiCl solution,46 dried

Blood without added NH4Cl produced a response significantly above thebaseline current, indicative of the ammonia level in the blood sample.Blood with added NH4Cl produced an approximately 2-fold higher response,with an incremental increase of about 20 nA. The results suggest thatthe glass fiber filter itself may be effectively alkalinizing the bloodsample to release ammonia, thus eliminating the need for treating thefilter with base. Most importantly, this preliminary experimentindicates that electrochemical detection of NH3 released from a drop ofblood on a solid support is feasible. By using a small samplecontainer/substrate 12 to match the small sample size, it may bepossible to provide both an increase in the response current and also asubstantial decrease in the time to reach a current plateau. Theseimprovements may make it possible to accurately quantify whole bloodammonia from a single drop of blood within seconds.

Although various embodiments and examples are described above, theseembodiments and examples should not be interpreted as limiting the scopeof the present invention. Any of a number of suitable modifications maybe made to any of the above-described embodiments, without departingfrom the scope. Thus, the embodiments are meant to be exemplary innature and not limiting.

What is claimed is:
 1. A method for measuring ammonia in a blood sample,the method comprising: positioning the blood sample in proximity with anammonia gas sensor; mixing the blood sample with an alkaline substance;generating a current with the ammonia gas sensor in response to ammoniagas released from the blood sample; and measuring the current generatedby the ammonia gas sensor, using a current measurement member coupledwith the ammonia gas sensor.
 2. A method as in claim 1, wherein theammonia gas sensor comprises an ammonia fuel cell, and whereinpositioning the blood sample comprises positioning the blood sample in asealed chamber that surrounds at least part of an anode end of theammonia fuel cell so that the anode end is exposed to the ammonia gasreleased from the blood sample.
 3. A method as in claim 2, whereinpositioning the blood sample further comprises attaching the bloodsample containment member to the ammonia fuel cell to form the sealedchamber.
 4. A method as in claim 3, wherein attaching the blood samplecontainment member to the ammonia fuel cell forms at least one aperturein the blood sample containment member to form the sealed chamber.
 5. Amethod as in claim 1, wherein the blood sample comprises no more thanabout 0.05 mL of blood.
 6. A method as in claim 1, wherein mixing theblood sample is performed before positioning the blood sample.
 7. Amethod as in claim 1, wherein mixing the blood sample is performed afterpositioning the blood sample.
 8. A method as in claim 1, wherein thealkaline substance is selected from the group consisting of: potassiumcarbonate; an aqueous solution of lithium chloride and lithiumhydroxide; other hydroxides; and other salts.
 9. A method as in claim 1,further comprising stirring or agitating the blood sample and thealkaline substance at least one of during or after the mixing step. 10.A method as in claim 1, wherein positioning the blood sample comprisespositioning a substrate holding the blood sample in proximity with ananode end of the ammonia gas sensor.
 11. A method as in claim 1, whereinmeasuring the current comprises measuring with an ammeter.
 12. A methodas in claim 1, wherein measuring the current comprises measuring with apotentiostat.
 13. A method as in claim 1, further comprising displayingthe measured current on a display member coupled with the currentmeasurement member.
 14. A method as in claim 1, wherein the method isperformed using a portable ammonia measurement device comprising a bloodsample containment member, an ammonia gas sensor coupled with the bloodsample containment member, and a current measurement member coupled withthe ammonia gas sensor, and wherein the portable ammonia measurementdevice is configured to be held in one hand of a user.
 15. A device formeasuring an ammonia level in a blood sample, the device comprising: ablood sample containment member; an ammonia gas sensor comprising anammonia fuel cell coupled with the blood sample containment member; anda current measurement member coupled with the ammonia gas sensor.
 16. Adevice as in claim 15, wherein the blood sample containment member isremovably coupled with the ammonia gas sensor.
 17. A device as in claim15, wherein the blood sample containment member comprises a compartmentfor holding the blood sample, wherein the compartment forms a sealedchamber when the blood sample containment member is coupled with theammonia gas sensor, and wherein the sealed chamber encloses an anode endof the ammonia gas sensor and the blood sample.
 18. A device as in claim17, wherein the current measurement member comprises an electric circuitcoupled at one end with the anode end of the ammonia gas sensor and atan another end with a cathode end of the ammonia gas sensor.
 19. Adevice as in claim 15, wherein the blood sample containment membercomprises a substrate for holding the blood sample.
 20. A device as inclaim 19, further comprising a substance pre-applied onto the substratebefore application of the blood sample to the substrate.
 21. A device asin claim 20, wherein the substance is selected from the group consistingof: potassium carbonate; an aqueous solution of lithium chloride andlithium hydroxide; other hydroxides; and other salts.
 22. A device as inclaim 15, wherein the current measurement member comprises an ammeter.23. A device as in claim 15, wherein the current measurement membercomprises a potentiostat.
 24. A device as in claim 15, furthercomprising a housing at least partially containing the ammonia gassensor and the current measurement member, wherein the blood samplecontainment member is removably couplable with the housing.
 25. A deviceas in claim 24, wherein the blood sample containment member comprises acontainer couplable with the housing to form a sealed chamber in whichthe blood sample is contained.
 26. A device as in claim 25, wherein theblood sample containment member comprises a sealed vial comprising amembrane, and wherein coupling the blood sample containment member withthe housing pierces the membrane and forms the sealed chamber.
 27. Adevice as in claim 24, wherein the blood containment member comprises asubstrate, and wherein the housing comprises a slot into which thesubstrate is advanced to expose the blood sample to the ammonia gassensor.
 28. A device as in claim 24, further comprising a display on thehousing for displaying the measured blood ammonia level.
 29. A device asin claim 28, wherein the display is configured to display at least oneof a numerical indication or a linear representation of the measuredblood ammonia level.
 30. A device as in claim 15, further comprising atwo-part housing, wherein a first part of the two-part housing at leastpartially contains the ammonia gas sensor and the current measurementmember, and wherein a second part of the two-part housing at leastpartially contains the blood sample containment member.
 31. A device asin claim 15, wherein the device is configured to measure the ammonialevel in the blood sample, wherein the blood sample comprises no morethan about 0.05 mL of blood.
 32. A device as in claim 15, wherein thedevice is sufficiently small to be held in one hand of a user while inuse.